Different cell populations appear as candidates for cell-based therapies, in particular in neonates: Hematopoietic stem cells (HSC), endothelial progenitor cells (EPC), mesenchymal stem cells (MSC) as well as regulatory T cells (Treg) exerting immune modulatory and pro-regenerative activities by direct cellular action or secretion of pro-regenerative, anti-apoptotic, immunomodulatory factors. The suppressive activity of Tregs from umbilical cord blood (UCB) has been attributed to the observed reduced severity of graft versus host disease (GvHD) in UCB transplantation (Tolar J, Hippen K L, Blazar B R. Immune regulatory cells in umbilical cord blood: T regulatory cells and mesenchymal stromal cells. Br J Haematol 2009 October; 147(2):200-6).
Mesenchymal stem/stromal cells (MSCs) have emerged as one of the most intensely studied adult stem cell population within the last 15 years (Bieback K, Brinkmann I. Mesenchymal stromal cells from human perinatal tissues: From biology to cell therapy. World J Stem Cells 2010 Aug. 26; 2(4):81-92). The therapeutic interest initially was based on their multi-lineage differentiation potential. However, it recently turned out that only few cells engraft and differentiate after either local or systemic delivery of MSCs in a number of different injury models. Subsequent to the detection of profound immune modulatory activities, MSCs were successfully applied to treat steroid-refractory GvHD (Le Blanc K, Rasmusson I, Sundberg B, Gotherstrom C, Hassan M, Uzunel M et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004 May 1; 363(9419):1439-41).
Since then, a number of different studies have addressed the impact of MSCs on GvHD with different outcomes. In most of these studies, the infusion of MSCs appeared to be safe. However, a positive impact on GvHD is still discussed controversially (Baron and Storb, 2011). Although MSCs can be raised from virtually every tissue within the human body, they are a very rare population comprising about 0.001 to 5% of cells in different tissues (Bieback K. Basic Biology of Mesenchymal Stem Cells. Transfus Med Hemother 2008; 35(3):151-2).
Exosomes are naturally occurring small membrane vesicles (80-160 nm) released by a huge variety of cell species. Containing a combination of lipids and proteins as well as RNAs, exosomes participate in intercellular communication processes. They can be isolated from all body fluids including blood plasma, urine, saliva, breast milk, bronchial lavage fluid, cerebral spinal fluid, amniotic fluid and malignant ascites (Ludwig A K, Giebel B. Exosomes: Small vesicles participating in intercellular communication. Int J Biochem Cell Biol 2011 Oct. 19).
Initially, exosomes were discovered in 1983 as small vesicles that are released by exocytosis upon fusion of so called multivesicular bodies with the plasma membrane. In 1996, exosomes were isolated from B lymphocytes, and were demonstrated to exhibit antigen-presenting characteristics. Since then, their role in immune-biological settings has been investigated in an increasing number of studies. It turned out that exosomes derived from mature dendritic cells for example exert immune-stimulatory functions, while tumor-derived exosomes often mediate immune-suppressive functions (Ludwig A K, Giebel B. Exosomes: Small vesicles participating in intercellular communication. Int J Biochem Cell Biol 2011 Oct. 19).
In 2010, Lai and co-workers provided evidence that exosomes released from MSCs mediate at least a proportion of the regenerative effects of clinically applied MSCs (Lai R C, Arslan F, Lee M M, Sze N S, Choo A, Chen T S et al. Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res 2010 May; 4(3):214-22). Since intravenously administered MSCs get rapidly trapped into the lung and are hardly found within infarcted hearts (Lee et al., 2009), the group of Lim searched for paracrine MSC effectors and showed in a first set of experiments that the supernatants of in vitro expanded MSCs are sufficient to mediate beneficial effects. Next, they purified exosomes from MSC supernatants and infused them in a murine myocardial ischemia model. Again, they observed a reduction of the infarct size (Lai et al., 2010).
Timmers et al. (in Timmers L, Lim S K, Arslan F, Armstrong J S, Hoefer I E, Doevendans P A, Piek J J, El Oakley R M, Choo A, Lee C N, Pasterkamp G, de Kleijn D P. Reduction of myocardial infarct size by human mesenchymal stem cell conditioned medium. Stem Cell Res. 2007 November; 1(2):129-37. Epub 2008 Mar. 8) describe the use of secretions of human MSCs as a therapeutic option in acute myocardial infarct. These MSCs are raised from human Embryonic Stem Cells (hESCs).
Fleissner et al. (in: Fleissner F, Goerzig Y, Haverich A, Thum T. Microvesicles as novel biomarkers and therapeutic targets in transplantation medicine. Am J Transplant. 2012 February; 12(2):289-97. Epub 2011 Nov. 14) describe microvesicles (MVs) including exosomes as emerging new biomarkers and potential regulators of inflammation and immunological processes. The particles contain proteins and genetic information including DNA and microRNAs that may be of importance for cell/cell communication. However, their role during and after organ transplantation and immunomodulatory effects is described to be only in its beginning of understanding. The authors speculate that MV modulation may emerge as a therapeutic approach in organ rejection therapy.
Horstman et al. (in: Horstman L L, Jy W, Minagar A, Bidot C J, Jimenez J J, Alexander J S, Ahn Y S. Cell-derived microparticles (MPs) and exosomes in neuroinflammatory disorders. Int Rev Neurobiol. 2007; 79:227-68) describe MPs and their possible roles in the pathophsyiology of some neuropathologies, especially those which are ischemic in nature, but probably also inflammatory ones. Tables include a listing of bioactive agents known to be carried on MP, many of which were heretofore considered strictly soluble, and some of which can be transferred from cell to cell via MP vectors, for example certain cytokine receptors.
Baron and Storb (in: Baron F, Storb R. Mensenchymal Stromal Cells: A New Tool against Graft-versus-Host Disease? Biol Blood Marrow Transplant 2011 Sep. 29) propose that immune modulatory activities of MSCs are thought to derive from secreted soluble factors rather than from direct intercellular interactions of engrafted MSCs with the patient's immune cells.
WO 2009/105044 describes a particle secreted by a mesenchymal stem cell and comprising at least one biological property of a mesenchymal stem cell. The biological property may comprise a biological activity of a mesenchymal stem cell conditioned medium (MSC-CM) such as cardioprotection or reduction of infarct size. The particle may comprise a vesicle or an exosome. The claims encompass exosomes obtainable from human embryonic stem cells derived from human embryos.
WO 2012/020308 is directed at compositions and methods for the treatment of tissue damage (e.g., acute or chronic) and related diseases, disorders or conditions based on the use of pathfinder cells, extracellular secretomes thereof, and/or pathfinder cell-associated micro RNAs (miRNAs). In some embodiments, the present invention provides a method for treating inflammation comprising a step of administering a population of cells, or extracellular secretomes thereof, to an individual suffering from a disease, disorder or condition characterized by inflammation of one or more tissues, wherein the cells are originated from an adult tissue and wherein the cells induce an anti-inflammatory response.
WO 2012/053976 discloses the use of exosomes that are secreted by mesenchymal stem cells in order to promote hair growth and wound healing. These effects are disclosed in connection with pro-inflammatory cytokines and proteases. WO 2012/053976 speculates about an immune-modulatory effect of the exosome-preparation.
WO 2012/020307 discloses the therapeutic use of microvesicles including exosomes in the treatment of inflammation and lesions of cerebral trauma. Similarly, WO 2012/087241 describes the use of exosomes for the treatment of the diseases as mentioned.
The currently proposed strategies in the prevention and/or therapy based on exosomes suffer from several disadvantages. Most problematic is their unimodal approach. This is in contrast to the biological complexity of the pathophysiology of the indications to be treated. Instead, it would be required to use a therapeutic cocktail that can interact with very different pathophysiological signaling cascades, and thus provide a more complete and effective prevention or treatment.
Mesenchymal stromal cells (MSCs) represent a heterogeneous subset of multipotent cells that can be isolated from several tissues including bone marrow and fat. MSCs exhibit immunomodulatory and anti-inflammatory properties that prompted their clinical use as prevention and/or treatment for severe graft-versus-host disease (GvHD). Although a number of phase III studies have suggested that MSC infusion was safe and might be effective for preventing or treating acute GvHD, definitive proof of their efficacy remains lacking thus far. Multicenter randomized studies are ongoing to more precisely assess the impact of MSC infusion on GvHD prevention/treatment, whereas further research is performed in vitro and in animal models with the aims of determining the best way to expand MSCs ex vivo as well as the most efficient dose and schedule of MSCs administration (see Baron and Storb, 2011). Although MSCs can be raised from virtually every tissue within the human body, they are a very rare population comprising about 0.001 to 5% of cells in different tissues. Especially UCB contains MSC at low frequency. Also, MSCs derived from human embryonic ESC meet ethical concerns.
Finally, exosomes that are obtained from MSCs of individual donors were found to be very heterogenic. Thus, it seems reasonable to assume that subtypes of populations of MSCs with quite different potential are present. Currently, this problem is completely unresolved.
It is therefore an object of the present invention to provide a safe and more effective preparation of exosomes that is suitable for the treatment of diseases and conditions that involve inflammatory reactions. Other objects of the present invention will become apparent to the person of skill upon studying the present description of the invention.
In a first aspect thereof, the above object is solved by a pharmaceutical preparation comprising exosomes, obtainable by a method comprising the following steps:
a) providing a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) comprising exosomes,
b) enriching said exosomes, optionally comprising polyethylene glycol precipitation,
c) determining an in vitro immunomodulatory effect, in particular an anti-inflammatory effect and/or immune suppressive effect, of said enriched exosomes by, for example, a reduced IL-1β, TNF-α, T-cell proliferation, and/or IFN-γ cytokine response of effector cells of a donor, andd) selecting those enriched exosomes that exhibit an immunomodulatory effect, in particular an anti-inflammatory effect and/or immune suppressive effect.
Considering exosomes as a novel, very potent tool in regenerative and in immune-modulatory therapies, the inventors have set up new purification strategies and technologies to enrich for and to analyze exosomes. Amongst other indications, the inventors evaluated their applicability for use in immune-modulatory (inflammatory) therapies, such as GvHD therapy, and already treated an otherwise treatment-resistant grade IV acute GvHD patient with MSC-derived exosomes. The GvHD symptoms of the patient decreased dramatically with the specific exosome therapy and the patient remained stable for five months.
Furthermore, in order to prove effectiveness of the prevention and therapy of neuronal damages, in particular in neonates, experiments with LPS confronted rats were performed that showed a clear positive reaction following the administration of the inventive pharmaceutical preparation.
Preferred is a pharmaceutical preparation according to the present invention, wherein said neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) comprising exosomes are selected from human MSCs, and preferably from human MSCs derived from umbilical cord blood, umbilical cord tissue, placenta, bone or adipose tissue. The stem cells of the present invention are strictly non-embryonic derived stem cells. Most preferably, the MSCs that have been cultured in the presence of platelet lysate, and the preparation can contain said lysate and/or fractions thereof.
The pharmaceutical preparation according to the present invention is specifically enriched for exosomes. For this, generally any suitable method for purifying and/or enriching can be used, such as methods comprising magnetic particles, filtration, dialysis, ultracentrifugation, ExoQuick™ (Systems Biosciences, CA, USA), and/or chromatography. Nevertheless, preferred is a method that comprises polyethylene glycol precipitation and/or chromatographically enrichment using the monolithic technology (e.g. CIM®, BIA separations, Austria) as stationary phases instead of columns packed with porous particles. Monoliths are continuous stationary phases that are cast as a homogeneous column in a single piece and prepared in various dimensions with agglomeration-type or fibrous microstructures. (see Iberer, G., Hahn, R., Jungbauer, A. LC-GC, 1999, 17, 998). Using these methods, surprisingly active fractions containing exosomes could be obtained.
Then, in order to identify the most suitable fraction according to the invention fractions being enriched with exosomes are tested for their in vitro immunomodulatory effect, in particular an anti-inflammatory effect and/or immune suppressive effect, and can further be analyzed, in microbiological, in virulence and in pyrogen tests to, for example, excluded possible contaminations. In addition, these fractions can be studied with regard to protein content, and particle size.
It could be found that fractions being enriched with exosomes were particularly useful in the methods according to the present invention, if they exhibited strong in vitro immunomodulatory effects in activity tests, where, following the addition of said exosome fraction, a reduced IL-1β, TNF-α and/or IFN-γ cytokine response of effector cells of a donor could be found. ELISpot assays showed that the IL-1β, TNF-α and/or IFN-γ cytokine response of effector cells are impaired towards allogeneic cells in the presence of exosome containing fractions. Other methods that could be used to test for in vitro immunomodulatory effects include, for example, Luminex, ELISA, and/or flow cytometry.
The present invention is thus based on the novel concept for an improved prevention and treatment of diseases, in particular in patients suffering from a having a risk of an inflammatory disease, neuronal disease, GvHD, stroke, and ischemia and associated complications, for example, for avoiding inflammatory reactions prior or during surgery, and the prevention of inflammatory conditions and reactions of patients that are connected to a life support machine. In one embodiment, the diseases can be selected from pre- or postnatal damages of the nervous system, such as for example, brain damages related to hypoxia, inflammation, and/or ischemia. In another embodiment, the diseases can be selected from graft-versus-host disease, or transplant rejections following organ transplantations, respectively.
In a particularly preferred embodiment of the invention, the exosomes-enriched fractions derived from adult mesenchymal stem cells (MSCs) that were enriched using a polyethylene glycol precipitation protocol, are prophylactically and/or therapeutically transfused into patients, in particular neonates and/or patients receiving transplants and/or patients undergoing surgery.
Preferably, the exosomes-enriched fractions are derived from MSCs that are selected from human adult MSCs, and preferably from human MSCs derived from umbilical cord blood, umbilical cord tissue, placenta, bone or adipose tissue, and MSCs that have been cultured in platelet lysate.
The pharmaceutical preparation according to the present invention preferably is enriched for exosomes that comprise biological factors, such as, for example, proteins, such as anti-inflammatory cytokines, IL-10, TGF-β1, and HLA-G, and/or nucleic acids, such as, for example, miRNAs. This leads to the further advantage according to the invention that a) a multimodal (complex) intervention is performed, b) biological physiological (“self”) substances are used, and c) unwanted side effects of the preparation are reduced.
The present invention constitutes a multimodal intervention, and thus not only a specific factor is used (and only a part of the cascade (or of the underlying clinical phenotype) would be intervened with), but biologically complex and endogenous mediators and modulators are used. These components are found in every human, and therefore no significant adverse side-effects are expected.
Preferred is a pharmaceutical preparation according to the present invention, wherein said levels of TGF-β1 as measured in the activity tests are at least ten times higher (at 60.5×108 particles/mL) in the preparation, preferably at least 20 times higher, than in plasma levels of healthy controls. Further preferred is a pharmaceutical preparation according to the present invention, wherein the numbers of IL-1β, TNF-α and/or IFN-γ producing PBMC were found to be reduced in said patient more than fifty percent after the last application (p<0.0001, One-way ANOVA), compared to the cytokines' responses before MSC-exosome administration.
Further preferred is a pharmaceutical preparation according to the present invention, wherein said exosomes have a size of between about 70 to 200 nm, preferably between about 70 to 140 nm, or more preferably between about 70 to 120 nm. “About” shall mean a +/−10% deviation. Further preferred, the exosomes are positive for cellular exosome markers, and even further preferred the protein content of the pharmaceutical preparation is higher than 1 mg/ml.
In another aspect of the present invention, the pharmaceutical preparation according to the present invention, is suitable for i.v. administration, such as for example, intravenous administration or infusion into a patient in need thereof.
Another aspect of the present invention then relates to a method for producing a pharmaceutical preparation according to the present invention, comprising the following steps: a) providing a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs), optionally from MSCs that have been cultured in platelet lysate, comprising exosomes, b) enriching said exosomes, optionally comprising polyethylene glycol precipitation, c) determining an in vitro immunomodulatory effect, in particular an anti-inflammatory effect and/or immune suppressive effect, of said enriched exosomes by, for example, a reduced IL-1β, TNF-α, T-cell proliferation, and/or IFN-γ cytokine response of effector cells of a donor, d) selecting those enriched exosomes that exhibit an immunomodulatory effect, in particular an anti-inflammatory effect and/or immune suppressive effect, and e) admixing said enriched exosomes of step d) with at least one suitable pharmaceutical excipient and/or carrier.
Preferred is a method for producing a pharmaceutical preparation according to the present invention, wherein said neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) comprising exosomes are selected from human MSCs, and preferably from human MSCs derived from umbilical cord blood, umbilical cord tissue, placenta, bone or adipose tissue. The stem cells of the present invention are strictly non-embryonic derived stem cells. Most preferably, the MSCs that have been cultured in the presence of platelet lysate, and the preparation can contain said lysate and/or fractions thereof.
The method for producing a pharmaceutical preparation according to the present invention comprises the step of specifically enriching for exosomes. For this, generally any suitable method for purifying and/or enriching can be used, such as methods comprising magnetic particles, filtration, dialysis, ultracentrifugation, ExoQuick™ (Systems Biosciences, CA, USA), and/or chromatography. Nevertheless, preferred is a method that comprises polyethylene glycol precipitation and/or a monolithic method (see above), since using these methods, surprisingly active fractions containing exosomes could be obtained.
Preferred is a method for producing a pharmaceutical preparation according to the present invention, wherein fractions that were enriched for exosomes are further analyzed in microbiological tests, virulence tests, protein content, pyrogen tests, and particle size, in order to identify the most suitable fraction according to the invention.
It could be found that fractions that were enriched for exosomes were particularly useful in the methods according to the present invention, if they exhibited strong in vitro immunomodulatory effects in activity tests, where upon the addition of said exosome fraction, a reduced IL-1β, TNF-α and/or IFN-γ cytokine response of effector cells of a donor could be found. Preferred is a method according to the present invention, wherein said levels of TGF-β1 as measured in the activity tests are at least ten (or at least twenty) times higher (at 60.5×108 particles/mL) in the preparation than in plasma levels of healthy controls. Further preferred is a method according to the present invention, wherein said exosomes have a size of between about 70 to 200 nm, preferably between about 70 to 140 nm, or more preferably between about 70 to 120 nm. “About” shall mean a+/−10% deviation. Further preferred, the exosomes are positive for cellular exosome markers, and even further preferred the protein content of the pharmaceutical preparation is higher than 1 mg/ml.
Furthermore, the method for producing a pharmaceutical preparation according to the present invention comprises d) admixing said enriched exosomes of step c) with at least one suitable pharmaceutical excipient and/or carrier. In general any suitable pharmaceutically acceptable excipient and/or carrier can be used. Preferably, said pharmaceutically acceptable excipient and/or carrier renders said preparation suitable for i.v. administration, such as for example, intravenous administration or infusion.
Yet another aspect of the present invention then relates to a pharmaceutical preparation comprising exosomes derived from a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs), preferably according to the present invention as described herein for use in the prevention and/or treatment of diseases.
Preferably, the diseases that are prevented and/or treated using the pharmaceutical preparation comprising exosomes derived from a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) according to the present invention are selected from the group consisting of inflammatory diseases, neuronal diseases, transplant rejections, GvHD, stroke, and ischemia.
Preferably, the diseases that are prevented and/or treated using the pharmaceutical preparation comprising exosomes derived from a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) according to the present invention are selected from pre- or postnatal damages of the nervous system, such as for example, brain damages related to hypoxia, inflammation, and/or ischemia.
Most preferably, the diseases that are prevented and/or treated using the pharmaceutical preparation comprising exosomes derived from a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) according to the present invention are selected from graft-versus-host disease, and transplant rejections following organ or bone marrow transplantation.
Yet another aspect of the present invention according to the present invention then relates to a method for preventing and/or the treatment of a disease selected from the group consisting of inflammatory diseases, neuronal diseases, transplant rejections, stroke, and ischemia in a patient, comprising administering to said patient an effective amount of a pharmaceutical preparation according to the present invention. Preferably, the disease that is prevented and/or treated is selected from pre- or postnatal damages of the nervous system, such as for example, brain damages related to hypoxia, inflammation, ischemia, graft-versus-host disease, and transplant rejections following organ transplantation.
Preferably, said preventing and/or the treatment according to the present invention comprises an administration through i.v. administration, such as for example, intravenous administration or infusion.
More preferred is a method for preventing and/or the treatment of a disease according to the present invention, wherein said patient is a newborn.
The invention thus also relates to the application of exosome-enriched fractions for the treatment of acquired neonatal as well as adult neural defects, for example the treatment of brain damages in neonates and grown-ups after hypoxia, inflammation, ischemia, etc. Furthermore, the treatment of ischemic strokes can be treated, and the adverse effect of the stroke can be reduced and/or reversed.
The invention thus also relates to the application of exosome-enriched fractions for the treatment of transplantation-related complications. Here, primarily the treatment of GvHD, and the rejection of solid organs following transplantation have to be named. Treatments can be both prophylactic and/or therapeutic.
Based on their proposed broad multi-lineage differentiation potential mesenchymal stem cells (MSCs) became one of the most intensively studied adult stem cell entities within the last 15 years. Up to now, more than 300 different NIH-registered studies have addressed potential therapeutic impacts of clinically administered MSCs in a variety of clinical settings including treatment of acute myocardial infarction, stroke and acute kidney failure.
Several studies showed that the vast majority of intravenously administered MSCs get rapidly trapped in the lung and are rarely recovered in other tissues. Therefore, MSCs might improve clinical outcomes by paracrine effects rather than by their previously proposed engraftment into damaged host tissues (Lee et al., 2009). Indeed, the group of Lim earlier showed that the supernatants of in vitro expanded embryonic stem cell derived MSCs contain small extracellular vesicles, so called exosomes, whose infusion is sufficient to mediate a reduction of myocardial infarction sizes (see Lim et al., 2010).